| Gegen-Qinlian decoction is a famous prescription by Zhang Zhongjing, an extraordinarily skilled practitioner in Chinese history. In this prescription, Radix Puerariae, the monarch herb, has the function of expelling heat from muscular and skin and clearing Yang of spleen and stomach to cure the diarrhea. Radix Scutellariae and Rhizoma Coptidis, the ministerial herbs, with bitterness taste and cold nature, can clear the interior heat and regulate intestine and stomach. Radic Glyeyrrhiazae, the adjuvant herb, mixes and regulates all of the herbs. The above four herbs work together as the prescription of relieving interior syndrome. It was proved by modern medical research that the decoction has the exactly effect on the diarrhea caused by rotavirus and bacteria. Chinese traditional medicines have the advantages of anti-virus. Gegen-Qinlian preparations have good effects on curing bacillary dysentery, chordapsus and infant viral diarrhea. The actions of the preparations on reducing fever and checking diarrhea are better than those of ordinary chemical drugs. The preparations of this kind of formula include tablet, small pellet,capsule and composition and so on. The small pellet and tablet of Gegen-Qinlian were written in China pharmacopoeia. Because the two preparations were approved as the new drugs in 1980s, there are some problems lying in the quality standards. It is necessary to develop the modem research to the formula.The active fraction preparation is composed of a series of active constituents extracted from the compound recipe, which always share the same mother nucleus or possess the same chemical characteristics. Thus it has the advantages of not only treating diseases through multiple targets but also controlling the quality of the preparation definitely. It is out of question that the active fraction preparation can improve the drug's safety, effectively and quality control standards.In this paper, the active fraction preparation of Gegen-Qinlian was studied with the standard decoction as the basis of the quality and pharmacodynamics. The contents included (1) the quality control of the raw materials in the preparation. (2) the quality control of the active constituents and fractions in the decoction. (3) the extraction and separation technology and chemical assay of the active fractions. (5) the comparison of chemical components in the decoction and active fractions by HPCE. (6) the studies on the preparation technology, quality standard and stability of the preparation and bioavailability of puerarin in the new preparation.1. Identification and quality control of the botanical raw materials in Gegen-Qinlian decoctionThe active constituents in the raw material has concerned with the material's quality. Although the constituents are complex, the ratio of the components was relatively fixed. Under the condition of guaranteeing the species and families of the botanical raw materials, the quality of the herbs can be ensured under the control of one or several particular constituents. In order to keep the consistency and ensure the quality of the preparation and provide the standard for the manufacture, the quality of the herbs was monitored by HPLC. It was confirmed that the contents of puerarin, baicalin, berberine hydrochloride and glycyrrhizic acid in Radix Puerariae, Radix Scutellariae, Rhizoma Coptidis, Radic Glyeyrrhiazae were in the range of 3.5-5.0%, 14.5-18.0%, 8.5-11.0% and 20.-2.5% respectively.2. Determination of the contents of the active constituents and fractions in the decoctionThe active constituents in the decoction include peurarin, daidzin, daidzein, baicalin, chrysin,wogonin, berberine, coptisine, jateorhizine, glycyrrhizic acid and so on. The active constituents can be classified as three active fractions, isofavonone, flavonoids and alkaloids. In order to ensure the active constituents and fractions in the preparation were equal to those in the decoction and provide the guidance for the manufacture procedure, the contents of the active components and fractions were determined.The contents of the active constituents in the decoction were determined by HPLC and puerarin, baicalin, berberine hydrochloride and glycyrrhizic acid was 0.35g, 0.53g, 0.23g and 0.08g per dosage respectively. The methods of determining the active fractions were newly established by means of alumina column chromatography and ultraviolet spectrometry individually. The contents of isoflavone, flavonoids and alkaloids were 0.55g, 1.12g and 0.50g per dosage.3. Studies on the extraction and purification technology of the active fractions3.1 Extraction of isoflavonone from Radix PuerariaeIsoflavone, one of the active fractions, have significant pharmacological effects such as relieving fever, spasmolysis and so on. The extraction technology of isoflavonone was optimized by orthogonal design with the four factors and three levels. The results showed Radix Puerariae was extracted in turn with ten times and eight times volume of 95% ethanol for three hours, the contents of peurarin were satisfactory. 3.2 Purification of isoflavononeThe purification of isoflavone was optimized by solvent dissolution and acid precipitation. First, the ethanol extract of Radix Puerariae was dissolved with ten times volume of 95% ethanol. Then, the solution was acidified with acetic acid to pH 2-3 and deposited for 30min. At last, the solution was extracted with ethyl acetate twice, the layer of ethyl acetate was evaporated in vacuum and the residue was desiccated. The average content of total isoflavone was 53.30%.3.3 Extraction of flavonoids from Radix Scutellariae, Rhizoma Coptidis, Radic GlyeyrrhiazaeThe contents of the main constituents in the other three kinds of herbs always influenced each other in the decoction. In order to maintain the fixed ratio of the components, the extract technology of fiavonoids was optimized by orthogonal design with berberine hydrochloride and bacalin as markers. The herbs were extracted twice with twelve times volume of water for one hour, the results were satisfactory.3.4 Purification of flavonoids from Radix Scutellariae, Rhizoma Coptidis, Radic GlyeyrrhiazaeOn the basis of the theory, the solubility of the components decreases in the acidic environments, the extract of water was concentrated to the one-seventh volume and acidified to pH 1-2. The acidified solution was kept at 60℃for one hour on the water bath and then deposited for 24 hours. The average content of total flavonoids and alkaloids was 35.26% and 15.32%.4. Studies on the chemical components of the active fractions4.1 Analysis of isoflavone of Radix PuerariaeThe isoflavone extract of Radix Puerariae was separated on a Zorbax SB-C18 analytical column by HPLC with methanol-water-acetonitrile as mobile phase at a flow rate of 0.6ml/min. The elute was detected at 200-400nm by DAD. The results implied that the isoflavone was the main constituents in the fraction and the content of puerarin amounted to 42.2%.4.2 Analysis of flavonoids of Radix ScutellariaeThe flavonoids extract of Radix Scutellariae was separated on a Zorbax SB-C18 analytical column by HPLC with methanol-0.2% phosphoric acid-water as mobile phase at a flow rate of 0.6ml/min. The elute was detected at 200-400nm by DAD. There were nine exclusive peaks of flavonoids and baicalin was the highest one, amounted to 56.4%.4.3 Analysis of alkaloids ofRhizoma CoptidisThe alkaloids eluted form alumina column was separated on a Zorbax SB-C18 analytical column by HPLC with (0.01mol/L KH2PO4+0.2% triethylamine)- acetonitrile-methanol as mobile phase at a flow rate of 0.6ml/min. The elute was detected at 200-400nm by DAD. The results showed that benzyl isoquinoline alkaloids were the main constituents in the alkaloids and berberine hydrochloride was 63.4%, the highest one. The next was palmatine.5. Comparison of the chemical components in the decoction and the new preparation by HPCEIn order to prove the reasonability of the recombining active fractions and investigate the relativity between the chemical constituents in the decoction and preparation, the fingerprints were established by HPCE. The results showed that the chromatograms of decoction and preparation were similar. The ratio of the most peak area was from 0.80 to 1.25, but No.3, 6, 7 was from 0.28 to 0.59 may be caused by the procedure of the extraction and separation.6.Studies on the technology of the preparationIn order to improve the solubility of active fractions in the preparation, the disintegration agent L-HPC, cross-linking CMC-Na, CMS-Na and cross-linking PVP were studied by the dissolution tests. The results revealed that the dissolution of puerarin reached 40%, 80% and above 85% in 5min, 10min and 30min individually with cross-linking PVP.7. Studies on the quality control of the preparation7.1 Qualitative identificationThe qualitative identification of the herbs in the preparation was performed by TLC and the results were satisfactory.7.2 Quantitative analysisThe quantitative methods of the active fractions were established by UV and the precision, reproducibility and stability were satisfactory. The contents of isoflavone, flavonoids and alkaloids in the preparation were 18mg, 37mg and 16mg per dosage. The four active constituents were determined by HPLC and the contents of puerarin, baicalin, berberinc hydrochloride and glycyrrhizic acid were 10mg, 30mg, 6mg and 2.4mg per dosage respectively.7.3 Studies on the dissolution rate of puerarinTo ensure the solubility of the active constituent in the preparation, the dissolution rate of puerarin in Radix Puerariae was limited. The dissolute puerarin was not less than 40% in 10min and 80% in 30min.8. Studies on the accelerating stability of the preparationAccording to the guidance of China Pharmacopoeia (2000 Ed), the stability tests of the preparation had performed under the condition of 40℃and RH 75% for six months. The contents and dissolution rates of puerarin were determined at different time. The results revealed that the preparation was stable.9. Studies on the bioavailability of puerarin in the preparationPuerarin, one of the main active components of Gegen-Qinlian decoction, has the effect of reducing fever. Therefore, the absorption of puerarin in vivo has the close relationship with the preparation's function. In order to evaluate the preparation's absorption and pharmacokinetics process, the bioavailability tests were established with Beagles dogs. The results showed that both Cmax and AUC of puerarin in the decoction group was higher than that of in the capsule group. Tmax of puerarin in the decoction and capsule group was 25.2min and 54.4min respectively. In a conclusion the absorption of puerarin in the decoction was better than that of in the capsule. The results of pharmacokinetics can provide the foundation for the technology improvement and formula adjustment.
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